A polyhydroxyalkanoate (hereinafter, occasionally referred to as a “PHA”) is a polyester-type organic polymer molecule produced by a variety of microorganisms. PHAs are biodegradable thermoplastic polymers and are also producible from renewable resources. Accordingly, some attempts have been made to industrially produce a PHA as an environmentally friendly material or biocompatible material for various industrial applications.
Currently, many microorganisms are known to accumulate PHAs as energy-storage substances therein. A representative example of a PHA is poly-3-hydroxybutyric acid (hereinafter, abbreviated as “P(3HB)”), which is a homopolymer of 3-hydroxybutyric acid (hereinafter, abbreviated as “3HB”). P(3HB) is a thermoplastic polymer and it is biodegradable in nature. Thus, it has drawn attention as an environmentally friendly plastic material. Because of its high crystallinity, however, P(3HB) is hard and fragile. The range of practical applications thereof is accordingly limited. In order to expand the range of applications, it is necessary to impart P(3HB) with flexibility.
To this end, a PHA copolymer comprising 3HB and 3-hydroxyvaleric acid (hereinafter, abbreviated as “3HV”) (hereinafter, such copolymer is abbreviated as “P(3HB-co-3HV)”) and a method for producing the same have been developed (Patent Document 1 and Patent Document 2). Since P(3HB-co-3HV) had higher flexibility than P(3HB), it was considered that the range of applications of P(3HB-co-3HV) would be extensive. In practice, however, an increased 3HV molar fraction in P(3HB-co-3HV) does not lead to desirable physical changes. In particular, the flexibility of P(3HB-co-3HV) has not been sufficiently improved so as to be processed in the form of, for example, films, sheets, or soft-type packaging containers. Accordingly, the application of this material is limited to hard-type molded products, such as shampoo bottles or disposable razor handles.
Also, a PHA copolymer comprising 3HB and 3-hydroxyhexanoic acid (hereinafter, abbreviated as “3HH”) (hereinafter, such copolymer is abbreviated as “P(3HB-co-3HH)”) and a method for producing the same have been studied in order to further enhance PHA flexibility (Patent Document 3 and Patent Document 4). In related literature, P(3HB-co-3HH) was produced by fermentation using a wild-type strain of Aeromonas caviae isolated from soil and fatty acid, such as oleic acid or palmitic acid, as a carbon source, and the 3HH composition ratio of the resulting P(3HB-co-3HH) was 15 mol % when oleic acid was used as a carbon source and 5 mol % when palmitic acid was used as a carbon source.
Physical properties of P(3HB-co-3HH) have also been studied (Non-Patent Document 1). In this study, A. caviae is cultured using, as a single carbon source, a fatty acid containing 12 or more carbon atoms, and P(3HB-co-3HH) having a 3HH composition ratio of 11 mol % to 19 mol % is produced by fermentation. As the 3HH composition ratio is increased, the hard and fragile properties of P(3HB-co-3HH), as is the case with those of P(3HB), are gradually changed into flexible properties, and such flexible properties are superior to those of P(3HB-co-3HV). By changing the 3HH composition ratio, accordingly, an extensive range of physical properties that are applicable to polymers ranging from hard to soft polymers can be imparted to P(3HB-co-3HH). Thus, P(3HB-co-3HV) having a low 3HH composition ratio can be used for a product requiring rigidity, such as a television chassis, and P(3HB-co-3HV) having a high 3HH composition ratio can be used for a product requiring flexibility, such as a film. That is, an extensive range of applications can be expected.
Also, the PHA productivity of a transformant produced using, as a host, Cupriavidus necator (C. necator) and a PHA synthase expression plasmid, such as pJRDEE32 or pJRDEE32d13, into which pJRD215 (ATCC 37533) and a polyester synthase gene, the (R)-specific enoyl-CoA hydratase gene, or the like has been introduced has been investigated (Patent Document 5 and Non-Patent Document 2). While the amount of the cells was as low as 4 g/l after culture, it was found that polymer productivity would be improved via modification of cell culture conditions involving the use of plant oils and fats as carbon sources. For example, the amount of the cells would increase to 45 g/l, the polymer content would increase to 62.5%, and the 3HH composition ratio would increase to 8.1 mol %. Thus, attempts aimed at improvement of the 3HH composition ratio or polymer productivity of P(3HB-co-3HH) via culture techniques have been made (Patent Document 6). In general, however, plasmids would be deleted during the bacterial growth cycle as a result of introduction of a foreign gene using a plasmid. This destabilizes the introduced gene. Accordingly, such technique is not suitable for industrial production.
Meanwhile, it was reported that the 3HH composition ratio had improved via incorporation of the (R)-specific enoyl-CoA hydratase gene into the chromosome DNA of C. necator (Patent Document 7 and Non-Patent Document 3). According to this report, a plurality of (R)-specific enoyl-CoA hydratase genes may be inserted into a pha operon region comprising the pha synthase gene of C. necator, so as to increase the 3HH composition ratio to 10.5 mol %.
As described above, physical properties of P(3HB-co-3HH) vary in accordance with the 3HH composition ratio, and it can be thus used for an extensive range of applications. In order to attain desirable physical properties, however, it is necessary to precisely regulate the 3HH composition ratio. At the industrial level, in addition, it is preferable that a plurality of microorganism species be prepared for process simplification, so that a plurality of types of P(3HB-co-3HH) having different 3HH composition ratios can be produced under the same culture conditions. In the past, however, no improvement aimed at precise regulation of the 3HH composition ratio has been achieved in the breeding of microorganisms producing P(3HB-co-3HH).